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Unit 2: RNA isolation - Practice Quiz

BTY555 — Biotechnology Laboratory-I 60 Questions
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1 What is the primary purpose of using the Trizol reagent in the laboratory?

Isolation of RNA from yeast cell using Trizol method Easy
A. Isolation of RNA
B. Separation of lipids only
C. Isolation of proteins only
D. Amplification of DNA

2 Which chemical is the main active component of Trizol reagent?

Isolation of RNA from yeast cell using Trizol method Easy
A. Ethanol only
B. Sodium hydroxide only
C. Guanidinium isothiocyanate and phenol
D. Sodium chloride only

3 What is the main function of guanidinium isothiocyanate in RNA isolation?

Isolation of RNA from yeast cell using Trizol method Easy
A. Denaturing proteins and inhibiting RNases
B. Precipitating DNA only
C. Staining the RNA
D. Adding phosphate groups

4 Which reagent is added to Trizol to induce phase separation?

Isolation of RNA from yeast cell using Trizol method Easy
A. Chloroform
B. Glycerol
C. Acetic acid
D. Glucose

5 After phase separation, in which phase is the RNA found?

Isolation of RNA from yeast cell using Trizol method Easy
A. Interphase
B. Upper aqueous phase
C. The pellet at the bottom
D. Lower organic phase

6 Which molecule is present in the organic (phenol-chloroform) phase after separation?

Isolation of RNA from yeast cell using Trizol method Easy
A. RNA
B. Free nucleotides
C. Water-soluble salts
D. Proteins

7 Which alcohol is commonly used to precipitate RNA from the aqueous phase?

Isolation of RNA from yeast cell using Trizol method Easy
A. Isopropanol
B. Butanol
C. Glycerol
D. Methanol

8 What concentration of ethanol is typically used to wash the RNA pellet?

Isolation of RNA from yeast cell using Trizol method Easy
A. 75% ethanol
B. 10% ethanol
C. 50% ethanol
D. 100% ethanol

9 Which enzyme is the major threat to RNA integrity during isolation?

Isolation of RNA from yeast cell using Trizol method Easy
A. Polymerase
B. RNase
C. Ligase
D. DNase

10 Which type of water is preferred for dissolving the final RNA pellet?

Isolation of RNA from yeast cell using Trizol method Easy
A. Tap water
B. Mineral water
C. DEPC-treated water
D. Distilled water with RNase

11 Why must yeast cells be disrupted before RNA extraction with Trizol?

Isolation of RNA from yeast cell using Trizol method Easy
A. To release intracellular RNA
B. To remove chloroform
C. To increase phenol activity
D. To add nucleotides

12 Which absorbance ratio is commonly used to assess RNA purity?

Isolation of RNA from yeast cell using Trizol method Easy
A.
B.
C.
D.

13 At which wavelength do nucleic acids like RNA absorb maximally?

Isolation of RNA from yeast cell using Trizol method Easy
A. 230 nm
B. 280 nm
C. 600 nm
D. 260 nm

14 A pure RNA sample typically has an ratio close to which value?

Isolation of RNA from yeast cell using Trizol method Easy
A. 2.0
B. 3.5
C. 0.5
D. 5.0

15 Why is chloroform not miscible with the aqueous portion during extraction?

Isolation of RNA from yeast cell using Trizol method Easy
A. It contains dissolved RNA
B. It forms hydrogen bonds with water
C. It is a non-polar organic solvent
D. It is highly polar

16 What is the role of centrifugation in the Trizol method?

Isolation of RNA from yeast cell using Trizol method Easy
A. To separate the phases
B. To precipitate proteins in RNA
C. To amplify RNA
D. To lyse the cells

17 The lower temperature used during Trizol RNA isolation mainly helps to:

Isolation of RNA from yeast cell using Trizol method Easy
A. Reduce RNA degradation
B. Melt the cell wall
C. Speed up phase mixing
D. Increase enzyme activity

18 Which precaution is important while working with RNA to avoid contamination?

Isolation of RNA from yeast cell using Trizol method Easy
A. Handling samples at high temperature
B. Skipping the ethanol wash
C. Wearing gloves and using RNase-free tubes
D. Using ordinary glassware only

19 The RNA pellet obtained after isopropanol precipitation usually appears as:

Isolation of RNA from yeast cell using Trizol method Easy
A. A green solution
B. A black solid
C. A small gel-like or white pellet
D. A clear gas

20 Trizol reagent is generally which colour?

Isolation of RNA from yeast cell using Trizol method Easy
A. Blue
B. Green
C. Pink/red
D. Colourless

21 During Trizol-based RNA isolation from yeast, phenol and guanidine isothiocyanate work together primarily to:

Isolation of RNA from yeast cell using Trizol method Medium
A. Simultaneously lyse cells and inhibit RNase activity
B. Digest genomic DNA enzymatically
C. Neutralize the pH of the lysate to 7.0
D. Precipitate proteins into the aqueous phase

22 Yeast cells require an additional step compared to animal cells before Trizol lysis is fully effective. This is mainly because yeast:

Isolation of RNA from yeast cell using Trizol method Medium
A. Contain higher lipid content in membranes
B. Lack ribosomal RNA
C. Possess a rigid cell wall that resists chemical lysis
D. Have no nuclear envelope

23 After adding chloroform and centrifuging a Trizol lysate, three layers form. RNA is recovered from the:

Isolation of RNA from yeast cell using Trizol method Medium
A. Lower pink organic phase
B. White interphase layer
C. Pellet at the tube bottom
D. Upper colorless aqueous phase

24 Isopropanol is added to the aqueous phase during Trizol RNA isolation in order to:

Isolation of RNA from yeast cell using Trizol method Medium
A. Denature contaminating proteins
B. Precipitate RNA from solution
C. Adjust the salt concentration
D. Dissolve residual phenol

25 A student obtains an ratio of 1.6 for their yeast RNA sample. This most likely indicates:

Isolation of RNA from yeast cell using Trizol method Medium
A. Excess salt in the sample
B. Pure, high-quality RNA
C. Degraded RNA fragments
D. Protein or phenol contamination

26 Why is 75% ethanol (rather than 100%) used to wash the RNA pellet in the Trizol protocol?

Isolation of RNA from yeast cell using Trizol method Medium
A. It inactivates RNases more effectively than 100%
B. It fully dissolves the RNA pellet for purification
C. It removes salts while keeping RNA precipitated
D. It precipitates any remaining DNA

27 If a student over-dries the RNA pellet before resuspension, the likely consequence is:

Isolation of RNA from yeast cell using Trizol method Medium
A. The RNA degrades due to residual RNases
B. The pellet becomes difficult to dissolve and RNA yield drops
C. The ratio increases sharply
D. The RNA becomes contaminated with protein

28 DEPC-treated water is used throughout RNA isolation primarily to:

Isolation of RNA from yeast cell using Trizol method Medium
A. Adjust the pH of solutions
B. Chelate divalent metal ions
C. Increase RNA solubility
D. Inactivate RNases in the water

29 A researcher wants to check RNA integrity after Trizol isolation using denaturing agarose gel electrophoresis. Intact total RNA from yeast should show:

Isolation of RNA from yeast cell using Trizol method Medium
A. A single band at high molecular weight
B. Two sharp bands corresponding to 18S and 25S rRNA
C. Multiple bands below 100 bp
D. A smear across all molecular weights

30 Which order of the following steps is correct for Trizol RNA isolation from yeast?

Isolation of RNA from yeast cell using Trizol method Medium
A. Isopropanol precipitation → lysis → chloroform → ethanol wash
B. Ethanol wash → chloroform → lysis → isopropanol precipitation
C. Chloroform → lysis → ethanol wash → isopropanol precipitation
D. Lysis → chloroform → isopropanol precipitation → ethanol wash

31 The main role of chloroform in the Trizol method is to:

Isolation of RNA from yeast cell using Trizol method Medium
A. Precipitate RNA directly
B. Promote separation into aqueous and organic phases
C. Denature RNases irreversibly
D. Lyse the yeast cell wall

32 If the aqueous phase is accidentally contaminated by drawing up some of the interphase, the isolated RNA is most likely to be contaminated with:

Isolation of RNA from yeast cell using Trizol method Medium
A. Pure protein with no nucleic acid
B. Ribosomal RNA only
C. Lipids only
D. Genomic DNA

33 To calculate RNA concentration from spectrophotometry, an of 1.0 corresponds to approximately:

Isolation of RNA from yeast cell using Trizol method Medium
A. of RNA
B. of RNA
C. of RNA
D. of RNA

34 Why must samples be kept cold and processing done quickly during yeast RNA isolation?

Isolation of RNA from yeast cell using Trizol method Medium
A. To increase isopropanol precipitation efficiency
B. To keep the cell wall intact for longer
C. To improve chloroform phase separation
D. To minimize RNase-mediated RNA degradation

35 A student adds too little chloroform relative to Trizol during extraction. The most likely outcome is:

Isolation of RNA from yeast cell using Trizol method Medium
A. Increased RNA yield with higher purity
B. Poor phase separation and RNA contamination
C. Precipitation of RNA at the interphase
D. Complete loss of RNA into the organic phase

36 Glycogen or another carrier is sometimes added before isopropanol precipitation to:

Isolation of RNA from yeast cell using Trizol method Medium
A. Dissolve residual phenol contamination
B. Improve recovery of low-abundance RNA
C. Speed up phase separation with chloroform
D. Inactivate RNases in the sample

37 An ratio of 1.0 for a Trizol RNA sample most commonly indicates carryover of:

Isolation of RNA from yeast cell using Trizol method Medium
A. Genomic DNA fragments
B. Protein from the interphase
C. Degraded ribosomal RNA
D. Guanidine or phenol contaminants

38 During which step are proteins primarily removed from the yeast RNA preparation?

Isolation of RNA from yeast cell using Trizol method Medium
A. Final resuspension in DEPC water
B. Chloroform phase separation, into organic phase and interphase
C. 75% ethanol wash of the pellet
D. Isopropanol precipitation of the aqueous phase

39 A researcher needs mRNA-only samples but Trizol yields total RNA. What additional step is required?

Isolation of RNA from yeast cell using Trizol method Medium
A. Oligo-dT selection of poly(A) tails
B. Increasing the isopropanol volume
C. A second chloroform extraction
D. DNase digestion of the sample

40 If downstream RT-PCR shows amplification even in a no-reverse-transcriptase control, the RNA sample most likely contains:

Isolation of RNA from yeast cell using Trizol method Medium
A. Excess phenol from extraction
B. Residual genomic DNA contamination
C. High salt from incomplete washing
D. Degraded RNA fragments

41 Yeast cells possess a rigid cell wall composed largely of -glucans and mannoproteins. Compared to isolating RNA from mammalian cells, why does the standard Trizol protocol often yield poor RNA recovery from intact yeast unless modified?

Isolation of RNA from yeast cell using Trizol method Hard
A. Yeast RNases are activated specifically by guanidinium isothiocyanate
B. The cell wall resists phenol-guanidinium penetration, preventing efficient lysis and RNA release
C. Trizol chemically degrades -glucans, releasing inhibitors that shear RNA
D. The mannoprotein layer binds RNA irreversibly during phase separation

42 During phase separation after chloroform addition, a student observes that the RNA-containing aqueous phase is unusually small and the interphase is thick and cloudy. Which factor most likely explains this observation?

Isolation of RNA from yeast cell using Trizol method Hard
A. Guanidinium was omitted, so no phase separation could occur
B. Too much starting biomass overwhelmed the Trizol volume, leaving proteins/debris at the interphase
C. Excess chloroform dissolved the RNA into the organic phase
D. The sample was incubated too long at room temperature before centrifugation

43 The acidic pH of Trizol (phenol at pH ~4.5) is central to selective RNA recovery. What would most likely happen if a neutral/alkaline phenol (pH ~8.0) were mistakenly used?

Isolation of RNA from yeast cell using Trizol method Hard
A. RNA would move into the organic phase and be lost
B. Proteins would fail to denature and clog the aqueous phase
C. The phases would fail to separate entirely
D. Both DNA and RNA would partition into the aqueous phase, contaminating the RNA

44 After isopropanol precipitation, a student measures while . What is the most accurate interpretation?

Isolation of RNA from yeast cell using Trizol method Hard
A. Protein contamination is low but residual guanidinium/phenol (chaotropic salts) remains
B. The sample contains predominantly DNA rather than RNA
C. The RNA is highly pure and ready for downstream use
D. The sample is heavily protein-contaminated

45 Why is 75% ethanol (rather than 100% ethanol) specified for washing the RNA pellet after isopropanol precipitation?

Isolation of RNA from yeast cell using Trizol method Hard
A. It dissolves genomic DNA selectively from the pellet
B. It denatures any co-precipitated RNases more effectively
C. It rehydrates the RNA to improve solubility later
D. It removes residual salts while keeping RNA precipitated and minimizing pellet loss

46 A researcher over-dries the RNA pellet under vacuum until it becomes glassy and transparent. What is the most likely consequence?

Isolation of RNA from yeast cell using Trizol method Hard
A. The pellet becomes difficult to redissolve, lowering effective yield and purity
B. Residual DNA precipitates onto the pellet
C. The ratio increases above 2.2
D. The RNA degrades rapidly due to heat

47 Glycogen or linear acrylamide is sometimes added as a co-precipitant during yeast RNA isolation. In what scenario is this most justified?

Isolation of RNA from yeast cell using Trizol method Hard
A. When DNA contamination must be removed selectively
B. When RNA concentration is very high, to prevent gel-like clumping
C. When RNA concentration is very low, to provide a visible carrier and improve recovery
D. When guanidinium levels are too high for precipitation

48 On a denaturing agarose gel, high-quality yeast total RNA typically shows two bright bands. If instead a smear with loss of sharp bands is observed, the most probable cause is:

Isolation of RNA from yeast cell using Trizol method Hard
A. Excess isopropanol during precipitation
B. RNase-mediated degradation during isolation
C. Incomplete removal of chloroform
D. Use of DEPC-treated water for resuspension

49 In yeast, the intact large and small ribosomal RNA subunits are approximately which sizes, and what ratio suggests good integrity?

Isolation of RNA from yeast cell using Trizol method Hard
A. 25S and 18S rRNA, with a ratio near
B. 28S and 18S rRNA, with a ratio near
C. 25S and 18S rRNA, with a ratio near
D. 23S and 16S rRNA, with a ratio near

50 A student wants to use isolated yeast RNA for RT-qPCR but detects genomic DNA amplification in no-RT controls. Given the Trizol acidic-phenol chemistry, what is the best corrective step?

Isolation of RNA from yeast cell using Trizol method Hard
A. Increase the chloroform volume to remove DNA
B. Repeat the isopropanol precipitation with more salt
C. Perform an on-column or in-solution DNase I treatment after RNA isolation
D. Wash the pellet with 100% ethanol instead of 75%

51 Why must all bead-beating or lysis steps for yeast RNA isolation with Trizol be kept cold (or in short bursts with cooling)?

Isolation of RNA from yeast cell using Trizol method Hard
A. To minimize heat-driven RNA degradation and RNase activity during mechanical disruption
B. To enhance the denaturation of genomic DNA
C. To prevent guanidinium from crystallizing out of solution
D. To increase phenol solubility and improve phase separation

52 If phase separation is performed and the aqueous phase is accidentally aspirated along with a portion of the interphase, the most likely downstream consequence is:

Isolation of RNA from yeast cell using Trizol method Hard
A. A falsely high ratio above 2.2
B. Complete loss of RNA into the organic phase
C. Protein and DNA carryover that lowers RNA purity
D. Precipitation failure due to insufficient salt

53 DEPC treatment of water and solutions is common in RNA work. Why must DEPC-treated water be autoclaved before use in Trizol-based protocols?

Isolation of RNA from yeast cell using Trizol method Hard
A. To decompose residual DEPC, which can otherwise carboxymethylate and inhibit RNA/enzymes
B. To sterilize it, since DEPC does not kill bacteria
C. To activate DEPC's RNase-inhibiting capacity
D. To remove dissolved oxygen that oxidizes RNA

54 A yeast RNA sample gives measured with a 1 cm path length and a 50-fold dilution. Using the RNA convention that corresponds to , what is the stock RNA concentration?

Isolation of RNA from yeast cell using Trizol method Hard
A.
B.
C.
D.

55 Compared with column-based silica kits, a key advantage of the Trizol method for yeast RNA that also becomes a limitation for automation is:

Isolation of RNA from yeast cell using Trizol method Hard
A. It inherently removes genomic DNA without any DNase step
B. It captures small RNAs and total RNA without size bias but requires hazardous phenol handling
C. It eliminates the need for cell wall disruption
D. It avoids all organic solvents while giving higher yields

56 During resuspension, a student notices the RNA pellet dissolves very slowly and the solution appears viscous. Which practice best resolves this while protecting RNA?

Isolation of RNA from yeast cell using Trizol method Hard
A. Adding a small volume of chloroform to reduce viscosity
B. Gentle warming to ~55–60 °C for a few minutes in RNase-free water with pipetting
C. Heating to 95 °C until fully dissolved
D. Vortexing vigorously at room temperature for several minutes

57 The chaotropic agent guanidinium isothiocyanate in Trizol contributes to RNA integrity primarily by:

Isolation of RNA from yeast cell using Trizol method Hard
A. Denaturing RNases and other proteins to prevent RNA degradation during lysis
B. Buffering the phenol phase to an alkaline pH
C. Selectively precipitating genomic DNA out of solution
D. Solubilizing lipids in the organic phase only

58 Two identical yeast cultures are processed, but one is harvested in log phase and the other in late stationary phase. The stationary-phase sample yields degraded, low-quality RNA. The most likely biological explanation is:

Isolation of RNA from yeast cell using Trizol method Hard
A. Trizol cannot lyse stationary-phase cells at all
B. Log-phase cells contain guanidinium-degrading enzymes
C. Stationary-phase cells have thicker walls and higher RNase/autolytic activity, complicating lysis and preserving RNA
D. Stationary cells lack ribosomes, so no rRNA can be recovered

59 Why is isopropanol, rather than a larger volume of ethanol, commonly used to precipitate RNA from the aqueous phase in the Trizol protocol?

Isolation of RNA from yeast cell using Trizol method Hard
A. Isopropanol precipitates nucleic acids at a lower volume, reducing tube volume and salt carryover
B. Isopropanol chemically removes residual phenol
C. Ethanol cannot precipitate RNA at all
D. Isopropanol selectively precipitates only mRNA

60 A troubleshooting log shows good RNA yield but consistently poor performance in downstream reverse transcription. Purity ratios are and . The most probable culprit inhibiting RT is:

Isolation of RNA from yeast cell using Trizol method Hard
A. Residual phenol/guanidinium carryover indicated by the low
B. Degraded RNA lacking intact templates
C. Genomic DNA competing for the enzyme
D. Protein contamination indicated by the value
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